SUMMARY Coronaviruses are sporadically emerging viruses responsible for SARS and MERS disease outbreaks. There are currently no direct treatments for these viruses, nor is there a vaccine which induces broad protection from infection. However, several stages in the virus life cycle are promising targets for therapeutic intervention. Cell entry is mediated by the large glycoprotein spike, which binds to host receptors and mediates fusion of the viral and host membranes. The ability of coronaviruses to adapt to new species or escape from the immune system is attributed to the viral spike protein. Once inside the cell, the viral RNA synthesis complex is assembled from 16 non-structural proteins (NSP) which transcribe, edit and modify viral RNAs and remodel ER membranes to create RNA replication factories. Expression of the viral structural proteins involves the RNA synthesis complex carrying out discontinuous strand synthesis to produce a nested set of viral mRNAs with truncations of the 5' open reading frames. Discontinuous strand synthesis is essential for the production of new virions and understanding its mechanisms will shed light on related viral processes such as viral recombination to generate spike variants with altered serotypes or host tropisms. During the K99 phase, I will obtain training in cryo-electron microscopy to complement my expertise in X-ray crystallography. I will use cryo-electron microscopy to examine the distinct conformations of the coronavirus spike protein as it binds host receptors and is primed for the fusion process by host proteases as I transition to the R00 phase of the award. These studies build on the recent structure determination of the HKU1-CoV spike protein from Dr. Andrew Ward's laboratory to which I contributed. Not only does this spike structure demonstrate the feasibility of the proposed experiments, but also provides a basis for new hypotheses of spike protein function. Also during the K99 phase, I will utilize the expertise of Dr. Erica Saphire's laboratory to develop RNA helicase assays to assess the function of the viral NSP13 helicase. I will use these assays to provide mechanistic, biochemical evidence to identify RNA templates upon which the NSP13 helicase stalls and may lead to induction of the RNA synthesis complex to carryout discontinuous strand synthesis. During the R00 phase, I will complement these studies with biochemistry, X-ray crystallography and cryo-electron microscopy to identify molecular mechanisms by which NSP13 recognizes RNA substrates and communicates with the RNA synthesis complex. These proposed studies will illuminate novel targets for antiviral therapy.